The present invention is directed to a quantitative testing apparatus and method which may be used for a wide range of diagnostic testing and evaluation of various cells, tissues, or other materials taken from the human body. The present invention is directed to an apparatus (hereinafter the "kit") and method used in image analysis using pattern recognition techniques to analyze and quantify cell constituents or components which may be stained. This kit and method are particularly useful and adaptable to the method and apparatus disclosed in application Ser. No. 794,937 filed Nov. 4, 1985, now U.S. Pat. No. 4,741,043 which is fully incorporated by reference herein. In a preferred embodiment the method and the kit may be used in the measurement of cellular DNA for the purpose of cancer diagnosis and prognosis.
As will be explained in greater detail, the present invention is directed to providing equipment of a user interactive nature for use not only by researchers, but also by a pathologist in a laboratory and to low-cost equipment which can be acquired by a typical pathologist laboratory.
The current state of the art in pathology laboratory is to estimate the content of cell constituents or components (hereinafter "cell objects") such as constituents or components of DNA by the visual observation of the pathologist who observes primarily the shape and texture of the cell objects after staining. For example, in connection with suspected cancer cells the pathologist observes the shape and texture of the cell objects and then classifies them into a normal category or into one of several abnormal cancer categories. These evaluations, however, are very subjective and can not differentiate and quantify small changes in DNA within individual cells or in very small populations of abnormal cells, which changes have clinical significance in the diagnosis and prognosis of cancer as discussed infra. Although there are commercially available general purpose flow cytometers, which are very expensive units and which can handle liquid blood specimens or tissue disaggregations, these cytometers are incapable of working on standard tissue sections and of using microscope slides which are the preferred specimen forms used in pathology laboratories. Additionally, an image analysis technique allows analysis of morphological features of cells such texture, in combination with size and shape of cell nuclei and alterations in nuclear-to-cytoplasmic ratios of cells whereas the flow cytometer does not allow such analysis.
The progression of the skill-of-the-art techniques for testing in anatomy, surgery, and histopathology has to date evolved to a primarily visual comparison of stain enhanced cells and tissues to human memory of previous examples. New advances in measurement, (i.e., the application Ser. No. 794,937) to quantify and replace these current state-of-the-art subjective comparisons to past memory, require calibration of the measurement instruments. Novel calibration means are required, e.g., as compared to chemical analysis calibration (where the state-of-the-art for calibrated measurement is well developed), because the material, although it is being read by light transmission measurements as in chemical analysis, is actually presented to the measurement instrument as a thin solid material, preserving cell and tissue morphology, on a transparent substrate. Methods, and the state-of-the-art techniques for calibrating such readings after suitable quantitative staining for specific cell or tissue parts, are essentially undeveloped and non-existent. Adequate calibration, such as described in this invention, will revolutionize and transform testing in these laboratories from subjective to objective. Such calibration preferably is on a test-by-test basis, i.e., on the individual microscope slide for each specimen, because the tested objects are so incredibly small, e.g., measuring picograms of DNA in cells with nuclei on the order of 100 micrometers.sup.2 in size, that very small shifts in light transmission or subtle staining variations make the measurement process too error prone without such calibration.
The use of image analysis generally requires staining cell objects on a microscopic slide. The use of image analysis techniques and equipment and stained specimens by pathologists in a conventional pathology laboratory involves solving a number of problems, including variation of stain, variation of optical densities of stained cell objects and calibration of microscopic slides with stained objects thereon, all of which have been overcome by the present invention. There are a number of available staining techniques which can be used. The Feulgen staining technique may be used to stain DNA in cell objects with dyes, for example, with thionin, Azure A, Azure C, pararosanilin and methylene blue. Proteins may be stained with congo red, eosin, an eosin/hematoxylin combination, or fast green. Enzymes may be made visible with diaminobenzidine or 3-amino-9 ethylcarbazole or alkaline phosphatase in combination with a dye substrate; cell organelles may be stained with methylene blue; and ribosomes with methylene blue and mitochrondia with giemsa stain. For purposes of this application "stain" includes an enzyme (such as alkaline phosphatase) in combination with a dye substrate to make something visible. Moreover, as used herein, stain includes counter stains such as methyl green. In breast cell cancer analysis some of these stains are used in combination with monoclonal antibodies which detect estrogen or progesterone receptors. Antigen analysis may include the steps of binding of monoclonal antibodies to the specimen and control cell objects. Later the monoclonal antibody may be conjugated with an enzyme stain. Also, the monoclonal antibody may be conjugated with a fluorescent material or stain. Then the fluorescent stain may be excited at a wave length to induce the fluorescence and then this may be observed at another wave length at which fluorescent emission occurs. When the antibody is made for a particular virus, the control cell specimen objects may be treated with a nucleic acid probe specific for the genome of the virus.
Variation in the degree of staining of cell objects and the variation of the optical density of the stained cell objects presents a problem in the quantitation of the stained cell objects through image analysis. The staining of cell objects, such as the DNA with Azure A, will vary substantially not only from slide to slide or from batch to batch by the same pathologist, but will vary substantially between different pathologists and different laboratories. Because the image analysis equipment is measuring grey level or optical densities and because it is desired to provide a true actual amount of DNA per cell in picograms from optical density measurements from stained cell objects, it is important to overcome the problem of different staining factors for different specimens. Also, image analysis techniques use microscopes and optical lighting which are adjustable to provide different intensities of light when used by the pathologist. Trained researchers, in research laboratories may be equipped to adjust the optical intensity to the desired conditions for image analysis by image pattern techniques, but this generally will not be accomplished with the precision necessary in the usual pathology laboratory. Thus, there is a need to overcome the problem of this optical density and staining variable.
Heretofore in cell analysis, an inexpensive and simple quantitation of cell objects has not been available. For example, except for those using only more expensive and sophisticated equipment, relative comparisons of data which are a function of cell object content have been only available to workers studying the proliferation of cell objects. In the case of DNA, absolute values of DNA content of cell nuclei in terms of picograms has not been readily available to laboratory workers for uses such as cell cycle analysis. Moreover in connection with DNA, this is clinically significant in the diagnosis and prognosis of cancer. The analysis of the DNA content of cells has been shown to be of value in the assessment of proliferations of benign and malignant cells. Abnormal DNA content (aneuploidy) has been observed consistently in numerous cancers such as prostate, colon, cervical, breast, and bladder. Also, preliminary data indicates that assessment of aneuploidy has prognostic value. See Atkin, Cytophotometric DNA Determination Corrrelated to Karyotype, Particularly Cancer, The International Academy of Cytology Analytical and Quantitative Cytology and Histology: 9:96-104 (1987). In addition the presence of an increased number of cells which are synthesizing DNA (so called "S phase" cells) has been shown to relate the extent of tumor cell proliferation, in some cases.
The method and kit of this invention coupled with any apparatus of carrying out image analysis of cell objects after staining, using pattern recognition techniques (such as the apparatus disclosed in Ser. No. 794,937, filed Nov. 4, 1985), permit a worker to readily and inexpensively not only detect minute alterations in cell objects including DNA, but also to measure and quantify the amount of cell objects as an aid to statistical analysis in research and patient diagnosis and treatment.
The present invention overcomes the problem of high costs heretofore associated with computerized equipment used for image analysis; and to this end, the present invention is an interactive system in which the pathologist performs a number of tasks including the selection, preparation, placement and staining of cells on microscopic slides. The pathologist is provided with the kit of the invention which includes stain and slides both of which are especially prepared and calibrated. The slide includes reference cells to aid in the diagnosis of the specimen cell objects and to assist in overcoming the staining density problem above-described. The present invention also permits location of cell objects for examination as to their morphology and preserves their location for a later analysis or corroborating analysis by a second pathologist when so desired. With respect to nuclei, measurements may be obtained as to area in microns, total nuclear optical density or nuclear mass in picograms, average nuclear optical density, nuclear texture, and deviation of the nuclear shape from being a round nucleus. Also, a number of such measurements may be made of the cell cytoplasm.
When the kit of this invention is used for cell analysis, tissue and cell specimens are applied to a slide which then is stained with a specific stain that combines proportionately with the cell objects which generally essentially renders invisible the remainder of the cell so that the image analysis measures the cell object content such as DNA which is concentrated principally at the nucleus of the cell. The stain associates with the cell object to provide a detailed nuclear structure and pattern which may be visually observed and interpreted by the pathologist using an apparatus for image analysis. In connection with DNA analyses for diagnosis and prognosis of cancer, the amount of DNA in the malignant cells generally is substantially greater than that for normal cells because the malignant cells usually are dividing and replicating rapidly or the malignant cells have abnormal numbers of chromosomes or have defective chromosomes.
The kit of the invention comprises a microscopic slide which includes a reference area and a specimen cell object area for receipt of specimen cells. The reference area contains a reference means for simultaneous staining for a predetermined time with the specimen cells or cell objects after the specimen cells or cell objects are applied to the specimen cell object area of the slide. According to the invention this simultaneous staining of the reference means and specimen cell objects with a stain of predetermined concentration permits a self-calibration of the slide as hereinafter described. The kit also includes one or more containers of stain and may include a container of rinse sulfonating agent for addition to a rinse used in preparation of the slide for microscopic image analysis. The amount of stain in the kit affects the optical density of the reference means and the specimen cell objects. This is an important aspect of the invention. After the staining the optical density of the reference means (and the specimen cell objects if they contain the material being investigated and measured such as DNA) will be a linear function of stain concentration per unit of material (such as stain concentration per cell object if the material being stained are cells) only over a select range of stain concentrations per stained cell object such that an optical density in the range of from about 0.1 to 0.8 is provided. Except for this linear portion, a curve of a plot of optical density versus stain concentration per cell will not be linear and/or not provide readily measurable or understood differences in optical densities with changes in stain concentrations per cell object. This is important to cell analysis. In cancer diagnosis and prognosis observation of varying DNA content by virtue of differing stain content and the resulting differing optical densities will be more readily detected and understood if the variation of optical density to stain concentration per cell is linear and the optical density is in the range of from about 0.1 to about 0.8. Quantitation of DNA, however, is only an example and analysis of any cell object by optical density and will be more readily understood if the optical density of the cell object is linear. Hence it is important that the stain in the kit be provided in an effective amount of stain to provide an optical density to the reference means after staining or a predetermined amount of time such that the optical density of the reference means will be a substantially linear function of the stain concentration of the reference means after staining and the optical density is in the range as aforesaid. The same is true of the specimen cell object if the object contains the material being referenced by the reference material.
The reference means for staining contains or constitutes any reference material which combines with stain proportionately to the combination of stain with the cell objects being analyzed. In connection with DNA analysis, the reference material may be rat liver nuclei, trout erythrocytes, chicken erythrocytes, dried DNA or cultured cell lines which reproduce themselves such as lymphoblastoid cells. In connection with proteins or enzymes the reference material may be any material containing a known amount of protein or enzymes to which an analysis is being directed.
The stain of the kit also may include a stain sulfonating agent. The stain sulfonating agent and rinse sulfonating agent are used in conjunction with acidic aqueous solutions of stain and rinse. Preparation of the slide frequently contemplates putting the stain in an acidic aqueous solution and then staining the reference means as well a the specimen cell objects with the aqueous stain solution. After the materials on the slide are stained, they are rinsed with a solution which also frequently is an acidic aqueous solution. In such cases consistent reproducible results demand stains and rinses having pHs within consistent relatively narrow ranges. A sulfonating agent which is compatible with the stain aids in binding the thionin or Azure A to the hydrolyzed DNA.
In an alternate embodiment of the invention, the microscopic slide of the kit includes an optical density reference area which area includes a material which has a predetermined known optical density to calibrate the microscopic slide with the instrument being used to study the specimen cell objects. Without the optical density reference area, the slide in conjunction with the kit described herein is self calibrating without regard to certain other variables that may change from analysis to analysis. These include variations in thickness and type of glass used in the slide as well as variations in temperature and humidity conditions encountered during analysis and which could affect analysis.
The method of the invention permits the quantitation of specimen cell objects by comparing the optical density of the stained specimen cell objects with the optical density of the stained reference material having known amounts of material to be quantified. For example, in connection with DNA quantitation, trout erythrocytes are known to have 5.6 picograms of DNA. Use of these cells as a reference material will permit the calculation of the DNA content of specimen cell objects in terms of absolute DNA weight when such specimen cell objects are simultaneously prepared and stained and the optical densities of the stained reference material and specimen cell objects are compared. This calculation and computer program relative thereto are described in my application Ser. No. PCT/US861/02409 filed Nov. 4, 1986 which application is fully incorporated herein. In breast cell analysis for the quantitation of estrogen receptors cultured breast cancer cells or tissue sections of organic material e.g. endometrium may be used as reference cells and as a source of reference cell objects.
In connection with the quantitation of nuclear DNA, the method of the invention includes providing a slide with a reference area and a specimen cell object area; providing a reference material in the reference area, the reference material having physical characteristics which include a known amount of DNA and permit association of the reference material with a stain which is proportional to an association of the stain with DNA; providing specimen cell objects in the specimen cell object area; simultaneously staining the reference material and the specimen cell objects with a stain in aqueous solution of stain for a predetermined amount of time, the stain in aqueous solution being in an effective amount to provide the reference material with an optical density after staining which will be a substantially linear function of stain concentration of the reference material; measuring the optical density of the reference materials after staining; measuring the optical density of the specimen cell objects after staining; and determining the quantitative amount of DNA in the specimen cell objects from the measured optical densities.
A very important alternate embodiment of the invention is rat liver cells nuclei as a reference means in conjunction with thionin stain. Rat liver has tetraploid cells having 13.4 picograms of DNA which give a number of measuring points for DNA content as well as diploid cells having 6.7 picograms of DNA. With tetraploid cells, rat liver tissue is advantageous over trout erythrocytes or other reference materials which include only large amounts of diploid cells, and hence, have fewer measuring points for DNA. After staining, the larger amounts of DNA in the rat liver nuclei, and the larger size of these nuclei, provide a reference material with an increased optical density. Larger size, more DNA and increased total optical density permits more precise calibration and thus less measuring error. Moreover, rat liver cells not only look substantially like human tissue, they combine with stain similar to the way human tissue combines with stain. This similarity results in similar optical densities for simultaneously stained human and rat tissue. Finally, rat liver tissue are readily cut, sectioned and applied to microscopic slides to provide a sample with an even distribution of cells which are oriented such that staining and observation are facilitated.
Thionin is important to the alternate embodiment and use of rat liver cells as a reference because it is very selective in staining DNA. Many other commercial stains such as Azure A often have impurities which will stain other material in a cell such as proteins. This is deleterious to the process of the invention because the relationship of optical density and the amount of DNA will not be as precise as with a pure stain which is precisely selective in staining DNA.
The kit and method of the invention permits an easy and inexpensive detection of minute alterations in specimen cell objects. In connection with cell object alterations in DNA content, this is done by providing a real and accurate measurement of the DNA in picograms. The invention also permits measurement and quantification of the amount of DNA and relates it to stored statistical analyses to aid in the diagnosis. More specifically, the invention in conjunction with my inventions disclosed and described in my applications Ser. No. 794,937 filed Nov. 4, 1985 and Ser. No. PCT/US86/02409 filed Nov. 4, 1986 in respect to DNA analysis allows an iterative analysis of specimen population cells and provides a histogram or display of the population distribution of the cells with respect to their DNA content and with respect to a standard DNA for normal cells so that subtle shifts in population distribution can be readily understood. To this end cell nuclei images are not only acquired and stored but the data therefrom can be integrated with statistical data to provide multi-variate analysis, discrimination of cells, histograms, and scattergrams of cells or cell populations.
Accordingly, a general object of the invention is to provide a new and improved apparatus and method for analyzing cells or other biological materials by using image analysis techniques.
A further object of the invention is to provide a new and improved kit which includes a stain and a slide or support for specimen cell objects which slide has a reference means or cell objects thereon wherein the stain with the slide permits calibration of the slide for image analysis of the slide in conjunction with image analysis equipment.
Another object of the invention is to provide a new and improved apparatus and method for making a ploidy analysis of cells using image pattern recognition equipment.
These and other objects and advantages of the invention will become apparent from the following description taken in connection with the accompanying drawings.